PROJECT SUMMARY Autism Spectrum Disorder (ASD) is a developmental disease that refers to a broad range of conditions characterized by challenges with sociability, repetitive behaviors, communication as well as cognitive deficits. Although the exact molecular mechanisms of ASD are poorly understood, cumulative evidence suggests that abnormal synapse development underlies many features of this disease. Most work in the field has focused on neuronal abnormalities while glial pathology in ASD has been overlooked for decades. A large number of autism-linked genes are highly expressed and significantly enriched in astrocytes suggesting a role for astrocyte dysfunction in the synaptic abnormalities observed in ASD. Astrocytes have been implicated in the pathogenesis of mouse models of syndromic ASDs. However, a detailed characterization of astrocyte dysfunction in ASD has not been completed, and it therefore remains unclear whether astrocyte dysfunction directly contributes to the synaptic plasticity and behavioral outcomes of ASD. Approximately 95 percent of ASD are idiopathic cases where autism is the primary diagnosis and not secondary to an existing condition caused by a well-known genetic variant. Human postmortem studies suggest that astrogliosis is one of the molecular features of ASD. However, it remains unclear whether astrocyte pathology plays a causative role in ASD, as opposed to representing a compensatory mechanism. The goal of this application is to understand the role of astrocyte dysfunction in idiopathic ASD. We have found that!mice exhibit social and memory deficits following neonatal brain engraftment of neural progenitors derived from ASD patient induced pluripotent stem cells. These progenitors terminally differentiated into astrocytes in transplanted brains, indicating that astrocytes account for the behavioral phenotypes observed in the ASD chimeric mice. Similarly, mice engrafted with astrocytes isolated from patient cerebral organoids exhibited reduced sociability. Based on the published literature and our preliminary studies, we hypothesize that astrocyte dysfunction is an underlying mechanism in ASD and contributes to specific behavioral impairments in this disorder. To test this hypothesis, we propose to determine 1) the behavioral impairments that astrocytes contribute to in ASD; 2) the functional deficits of ASD astrocytes (e.g., by assessing Ca2+ activity, protein profile and neurotransmitter uptake ability); and 3) the effects of ASD astrocytes on neurons (e.g., assessing neuronal network connectivity and synaptic plasticity). We will use a combination of techniques including patient-derived cerebral organoids, cell transplantation, two-photon Ca2+ imaging, electrophysiology and behavioral assays. Despite the growing realization of the importance of astrocytes in synaptic function and connectivity, astrocyte dysfunction represents a relatively unexplored mechanism for the onset and progression of ASD. The successful completion of this research will provide a coherent view of astrocyte involvement in ASD and potentially revolutionize our understanding of ASD pathogenesis and its treatment.